Process for culturing anaerobic bacteria



United States Patent O PROCESS FOR CULTURING ANAEROBIC BACTERIA GeorgeA. Jelfreys, Salem, Va.

No Drawing. Application February 11, 1953, Serial No. 336,446

11 Claims. (Cl. 195 -96) This invention relates to a new process forculturing obligate anaerobic bacteria in combination with an aerobicmicroorganism in the presence of air.

This application is a continuation-impart of application Serial Number126,437, filed November 9, 1949.

It is well known that many anaerobic bacteria produce growth promotingsubstances, B-complex vitamins, antibiotics and enzymes of economic andindustrial importance in many spheres of activity. Such beneficialfactors are produced by anaerobic bacteria in the rumen of herbivorousanimals, in cow manure, in hen feces, in the soil, etc. This has beendemonstrated by a number of research workers such as Ruben and Bird,Poultry Science, vol. XXVI, No. 5, 439; Ruben, Bird and Rothchild,Poultry Science, vol. XXV, No. 5, 526; McElroy and Goss, Journal ofNutrition, vol. 20, 527, 541; Hodge, Hanson and Allgeier, Industrial andEngineering Chemistry, vol. 44, No. 1, 132-35. Not only are thebeneficial substances produced by the anaerobic organisms important butfrequently, it is desirable to have available preserved cultures of highviable bacterial count.

The culturing of obligate anaerobic bacteria particularly andsemi-anaerobic bacteria to a somewhat lesser extent, even on alaboratory scale poses serious problems. On a commercial scale, the costis almost prohibitive. Obligate anaerobic bacteria must be grown out ofcontact with oxygen. The process most commonly employed both in thelaboratory and commercially is the submerged liquid method where theorganisms are grown in a broth in sealed tanks under anaerobicconditions. Great care must be exercised to maintain sterile conditionssince the accidental admission of contaminating microorganisms generallydestroys the broth culture in as much as the contaminant usually becomespredominant. Aside from the great skill and care required, operations ona commercial scale are very costly since they require large, expensivetanks and accessory equipment for sterilizing and maintaining anacrboicand sterile conditions.

Various methods for producing and maintaining anaerobic conditions havebeen proposed, the one most gen erally in use being replacement of airwith an inert gas such as carbon dioxide or nitrogen. Other suggestedmethods, which can be used only on a laboratory scale, include the useof a reducing chemical which combines with, and thereby removes, theoxygen in the air and introduction into the sealed propagating vessel ofan isolated culture of aerobic organisms which remove the oxygen toproduce the desired anaerobic conditions and which, after using up theavailable oxygen in the closed vessel, die off or otherwise becomedormant and inactive.

Although semi-anaerobic bacteria will grow to some extent under aerobicconditions, the rate of growth is very slow and for the production ofdesired factors such as enzymes, vitamins, etc., in any appreciableamounts, they must be grown anaerobically. It will be understood thatwhile my process is particularly directed to the culturing of obligateanaerobes, it is also effective for propagating semi-anaerobes.

2,766,176 Patented Oct. 9, 1956 Aside from the technical difiicultiesexperienced in propagating obligate anaerobic bacteria, it has hithertobeen impossible to produce highly concentrated cultures. The bacterialconcentration is generally in the order of several million per ml. orgm. and in rare instances may be as high as about million per gram.Obviously the higher the rate of gnowth, the larger are the amounts ofdesirable factors produced and the more potent are the cultures producedfor inoculation, feeding or other purposes.

Another difliculty hitherto has been the necessity for preserving theobligate anaerobic cultures under anaerobic conditions. This hasgenerally been accomplished by the lyophile process which involvesplacing the bacteria dispersed on agar or in serum in glass vials,quickly freezing by immersing the vials in a freezing solution at -20 to40 C., desiccating under high vacuum and sealing under vacuum. Thismethod is inordinately expensive and, because of its limiting factors,precludes its use for large scale production.

The object of this invention is to provide a process for culturingobligate anaerobic bacteria in synergistic relationship with activeaerobic microorganisms in the presence of air.

Another object is to provide a process for culturing obligate anaerobicbacteria which does not require special or costly procedures forproducing and maintaining sterile and anaerobic conditions and which, infact, may be cultured conveniently and economically in ordinaryincubators on a large, commercial scale.

Another object is to provide a desiccated viable culture of obligateanaerobic bacteria in admixture with active aerobic microorganisms inwhich the viable anaerobes are present in considerably higherconcentrations than has hitherto been generally obtained.

Another object is to provide cultures of obligate anaerobic bacteria andactive aerobic microorganisms which can be desiccated and preserved inthe presence of air by ordinary convenient methods without appreciableloss of viability for a long period of time.

Another object is to provide a preserved viable bacterial productcontaining high concentrations of desirable factors such as B-complexvitamins, growth factors, antibiotics, enzymes and the like for use infortifying livestock feeds and enriching food products for humanconsumption.

Still another object is to provide preserved cultures containing viableaerobic and obligate anaerobic microorganisms for digesting materialssuch as sewage and other industrial wastes, for soil inoculation and forthe production of soil conditioners and fertilizers such as composts.

Other objects and advantages will become obvious from the followingdetailed description.

In general my process comprises the culture of anaerobic bacteria inintimate relationship with active obligate aerobic vegetativemicroorganisms such as bacteria and molds on a nutrient substrate in thepresence of air. The actively growing aerobes provide the requisiteanaerobic conditions for the growth and propagation of the anaerobicorganisms. The relationship between the anaerobic and aerobic organismsis synergistic and in the case of the molds is symbiotic as well withconsequent unexpectedly high rate of growth and proliferation of theanaerobes and markedly high production of desired associated factors.The culture may be preserved by desiccating by ordinary methods in thepresence of air to produce a dry product containing a high concentrationof viable microorganisms, both anaerobic and aerobic, and associated,desired factors, such as B-complex vitamins, growth stimu latingsubstances, antibiotics, enzymes and the like.

The culture medium employed in my invention is preferably a granularsubstrate composed of Wheat bran or similar grain coating or vegetativefibrous material such as straw, hay or hulls of grain. To this is addedother essential nutrients such as proteinaceous materials or othernitrogenous equivalents, carbohydrates, trace minerals and inorganicsalts.

The composition of the culture medium is not critical, it beingessentlalonly to introduce the necessary food elements preferably inamounts for optimum growth. To some extent the composition will, ofcourse, be varied depending upon the particular organisms beingpropagated and other factors. In general a highly satisfactoryformulattion comprises about 60% to 85% of wheat bran or othervegetative, fibrous material, preferably cut up or ground to the sameparticle size as bran. A source of carbohydrate such as starch, dextroseor the like may be incorporated in a quantity preferably between aboutto 20% depending on the amount available in the particular fibrousmaterial employed. The proteinaceous material is desirably introduced inamounts between about 2 to 15% and may comprise any suitable source suchas vegetable protein meals as, for example, soy bean meal, or animalproteins as, for example, fishmeal, livermeal, meat meal, milk and thelike. Equivalent, assimi'lable non-proteinaceous nitrogenous materials,such as urea or ammonium salts as, for example, ammonium sulfate andammonium phosphate, may be used in place of, but preferably tosupplement the proteinaceous nutrient.

Small amounts of trace minerals such as cobalt, iodine, copper andmanganese are also desirable both to facilitate growth of the organismsand to increase the production of vitamin B12 and other growth factors.If the fibrous component is low in potassium content, 'it may besupplemented by the addition of monoand di'bas'ic potassium salts, forexample in amounts of about 0.25 to 0.5%.

Although not essential, it is desirable in some cases to incorporateorganic nutrients or chemicals that reduce the oxidation/reductionpotential to a minus condition. Examples of such organic materials aretomato juice, grasses and legumes or their juices, milk or milkproducts, ascorbic acid, sodium thioglycollate, sodium bisulfide, or anyvegetable or compatible chemical that has a tendency to produce a minusoxidation/ reduction potential. These reducing materials may be addedeither to the nutrient substrate or to the inoculating liquid or toboth. "Solid additives are preferably incorporated into the-solidsubstrate.

Water is added to the mixture of solid nutrients and substrate,desirably in amount comprising about 25% to 75% by weight. The mixtureis acidified to a pH of about 3.5 to 4.5 by the addition of HCl or othersuitable acid. The acidifying material is conveniently added to theWater prior -'to inclusion into the solid components. Acidity of thenutrient medium within this range of pH is desirable for more effectivesterilization.

The mixture is cooked and sterilized, as for example, with steam attemperatures preferably in the range of about 180 to 252 F. Theparticular temperature employed is determined by the type of cookingequipment employed, the length of heating time and the quantity ofmaterial. in general, only a few minutes are required at the highertemperatures and up to about 30 minutes at the lower temperatures withinthis range.

After sterilization, the nutrient medium is neutralized to a pH. ofabout 6.0 to 7.0, preferably about 6.5, with any suitable alkali such aslime or calcium carbonate.

The obligate anaerobic bacteria may be obtained'from any desired sourcedepending, of course, on the particular use to which the culture is tobe put and the associated factors desired. For example, where highconcentrations of B-complex vitamins, growth promoting substances,digestive enzymes and antibiotics are desired, the organ isms present inthe lumen of a ruminant, hen feces, cow manure and in certain soils areparticularly suitable.

The use of rumen organisms is particularly desirable where the productis to be employed as a livestock feed supplement not only because of thelarge quantities of beneficial factors produced but also because itprovides highly potent concentrations of rumen organisms for seeding andfortifying the rumen of ruminant livestock.

Where the product is to be used for the digestion of organic matter as,for example, in the treatment of sewage, the organisms may be ta-kenfrom septic tanks, trickling filters, activated sludge and the like. Forsoil conditioning, compost formation and the like suitable organisms maybe obtained from soil and humus.

Care should be exercised in obtaining the inoculating specimens to avoidcontamination. Rumen material, for example, is preferably taken from ahealthy steer or cow by means of a stomach pump under aseptic conditionsalthough the rumen contents obtained from freshly slaughtered, inspectedanimals is also utilizable. Similarly, aseptic conditions should beobserved in taking material from other sources such as hen feces, cowmanure, soil, etc. Samples from sewage sludge digestors should be takenfrom beneath the surface of the liquid.

The inoculating solution may be prepared by suspending the material perse containing the desired anaero'bes, such as the ground rumen contents,in water in which case all of the different types and varieties ofbacteria are given an opportunity to proliferate, or predominant strainsof bacteria may be isolated and subcultured in broth according to theusual methods well known to the art. The inoculating suspension orsubculture should be kept refrigerated under anaerobic conditions untilready for use. Preferably, it should be used within 48 hours but sampleshave been kept successfully for as long as a week. As a general rule,aqueous inoculating solutions or suspensions containing from 0.25 to 1%of bacterial material is sufficient for my purpose. As afore mentioned,it is frequently helpfulto introduce into the inoculating'liquid suchoxygen reducing materials as tomate juice, juices of legumes andgrasses, ascorbic acid, sodium thioglycollate, as for example in amountsof 0.01 to 0.1%, sodium 'bisulfide, as for example in amounts of 0.001to 0.01%.

The aerobic vegetative microorganisms may be either molds orbacteriawhich grow rapidly and require abundant oxygen. The molds may be, forexample, beneficial members of Aspergillus, Penicillium, Rhizopus,Mucor, Tric'hoderma .and similar types which include various beneficialyeasts. Molds which produce antibiotic substances, such asPenicillium,Aspergillus and the like are particularly efificacious since theyinhibit the development of undesirable organ-isms. i

The mold culture may include spores only or both spores and mycelium.

The aerobic bacteria should not be acid-forming and should be capable ofmaintaining a minimum pH of about 5.5 and preferably a minimum of about6.5 and ranging up to an alkaline pH, as for example about 7.5 to 8.Though not essential, it is preferable to employ bacterial strains andspecies which are also able to produce B vitamins and antibioticsubstances since, aside from the fact that this serves to increase theconcentration of these desired factors, the antibiotic substances helpto inhibit the growth of undesirable types of organisms in the substrateand to encourage the growth of those organisms which producegrowth-promoting factors.

Any beneficial aerobic bacteria which fulfill the aforedescribedconditions may be employed for my purpose. Particularly suitable aremembers of the genus Bacillus, family Bacillaceae. The mostdesirablespecies, for example are various strains of B. subtilis such as B.licheniformis which produces bacitracin, B. mesentericus, B. megalheriumor any beneficial species of this type which produces :B vitamins,antibiotic substances or enzymes.

The amount of mold or aerobic bacteria culture used may vary and, ingeneral, about 0.25% to 2% based on the'bran-substrate is-adequate. Theculture can beadded directly to the fibrous substrate or it can beintroduced into the water suspension of the anaerobic bacteria or bothmethods can be used.

The inoculating suspension or culture containing the anaerobic organismsis introduced into the sterilized granular nutrient substrate preferablyat a temperature of about 90 F. to 100 F. The mixing is preferably-doneunder limited aerobic conditions or under an inert gas.- A sufiicientamount of inoculum is added tomake a moist mixture, as for example, onecontaining about 70 to 125 parts of water per 100 parts of substratesolids.

The inoculated culture medium is spread on trays to form a layer about 1to-2 inches thick andthen placed in suitable incubators wheretemperature and humidity may be controlled with standard equipment. Thetemperature of the substrate should be maintained at a level whichstimulates active growth. In general, a range of about 82 to 105 isoptimum and, in most cases, it is well not to permit the temperature torise much above the maximum figure. Humidity should be maintained at ahi h level, preferably at about 80 to 90 percent relative humidity.

At the start of incubation only sufiicient air is admitted to supply therequirements of the aerobic microorganisms present. The amount of airintroduced should be increased to supply the increased requirements ofthe growing and proliferating aerobic organisms. The limited aerobicconditions may be readily controlled, as for example, by regulating airintake by diluting the air with an inert gas, such as nitrogen or CO2,in adjusted amounts to provide for optimum growth of both the aerobicand anaerobic organisms.

Within a period generally of about 2 to 6 hoursthe aerobic mold orbacteria begin to grow actively and in so.

doing produce an oxidation-reduction potential on the negative sidewhich is conducive to the growth of the an: aerobic bacteria. This isaccomplished by virtue of the fact that the aerobic organisms absorboxygen and excrete CO2 and other gases which, in effect, produces ananaerw obic environment in the vicinity of the anaerobes. The physicalgrowth and proliferation of the aerobes develop a protective solid mat,as, for example, the mold mycelium, around the anaerobes which furtherserves to exclude air.

After active growth and development of the aerobic organisms have becomewell established, it is generally no longer necessary to limit airintake into the incubating chamber although this may be continued tosome extent so long as it serves to provide optimum conditions foractive propagation of the anaerobes. It should be understood that tomaintain the necessary active growth of the aerobic organisms, anadequate supply of oxygen must be provided.

The active growth and respiration processes of the organisms generateheat and to keep the temperature from becoming excessive, it frequentlybecomes necessary to remove surplus heat. This may be accomplished bycutting off the external heating mechanism and increasing the movementof air over the outer surface of the material.

Maximum growth generally is attained in about 24 to 36 hours. Theculture may now be broken up and dried by subjecting it to moderatelyhot dry air according to conventional desiccating methods. If it isdesired to keep the organisms viable, the temperatureof the drying airshould be regulated so that the temperature of the culture itself doesnot exceed the thermal death point of the microorganisms. In most casesthe temperature of theculture should not be permitted to rise aboveabout 107 F. Drying temperatures may be higher if the only requirementis to obtain the beneficial factors produced by the organisms duringculturing. As drying proceeds, the anaerobic bacteria are protected bythe enveloping fibrous substrate and the aerobic microorganisms. 'It isalso believed that various end products produced in the culture act asbuffering and reducing agents to provide an oxidation-reductionpotential on the negative side which helps preserve the anaerobicbacteria. It will be understood, of course, that any other suitablemethod for desiccating the bacteria may also be employed.

The desiccated cultures obtained as afo'redescribedge'nerally contain atleast 2 billion viable obligate anaerobes per ml. or gram. The anaerobicbacterial count'is usually in the negihborhood of about 3 to 10 billionand in many instances is as high as 30 to 40 billion. The aerobic moldor bacteria count in the preserved cultures is proportionately high,generally being in the neighborhood of 1 or more billion. The desiccatedcultures, furthermore, remain viable for an indefinite period of time. I

It is obvious from the amazingly high obligate anaerobic bacterialcounts obtained that the active aerobic organisms provide an entirelyadequate anaerobic environment. However, this in itself does notcompletely explain the remarkable growth of the anaerobic bacteria.There is a synergistic relationship betwen the two types of organismswhich results in an acceleration both of growth and the production ofbeneficial substances such as B- complex vitamins, antibiotics, growthpromoting factors, enzymes and the like. These beneficial substances aredeveloped in considerably higher amounts than would normally be obtainedfrom the organisms grown separately. There is also, apparently, asymbiotic relationship between the anaerobic bacteria and molds when thelatter are employed as the aerobic organisms.

The synergistic effect is at least partially explainedby digestion ofthe substrate nutrients by each type of organism to form products whichare more readily assimilated by the other type of organism and by thesecretion of beneficial associated factors which either directly orindirectly stimulate growth of the other organisms.

EXAMPLE I A substrate composed of the following ingredients wasprepared:

950 lbs. wheat bran 5 lbs. dry brewers yeast 12 lbs. soybean oil meal 15lbs. whey 2 /2 lbs. mono calcium phosphate /2 lb. trace mineral mixture500 lbs. water 2500 mls. commercial H01 The above ingredients were mixedand then steamed in' an open mixer to a temperature of 200 F. and heldat' this temperature fifteen minues.

The mixture was cooled to 160 F.' at which time a mixture of equal partsof lime and calcium carbonate were added in sufiicient amounts to raisethe pH to 6.5.

The culture medium was then cooled to about F.

and inoculated with 200 lbs. of inoculating water prepared as follows: 1

Into a sterile kettle was added the following:

250 lbs. warm clean water 5 lbs. mold culture of. Aspergillus oryza 1lb. rumen contents obtained under aseptic conditions The mass was mixedfor not more than 1 /2 minutes under limited aerobic conditions and thenimmediately spread on trays in a layer about 1% inches deep and placedin a warm humid incubator of conventional type. The temperature of thematerial was maintained at 86 by live steam for a period of 20 hours.Until the 8th hour the air entering the incubator was diluted with CO,

zenith and at the 28th hour, the culture was broken up and dryinginstituted. Desiccation was accomplished A nutrient, fibrous substratewas prepared and processed as disclosed in Example I. lbs. of a moldculture of Aspergilhzs oryzae was employed as the aerobic or: ganism.The anaerobic organisms were contained in a sample taken directly from asewage sludge digestor, the percent-age used being 0.5% of theinoculating water.

Except that the temperature of incubation was maintained at about 82 F.or slightly lower than for rumen bacteria, an incubation procedure wasemployed as in Example I. At the end of the incubation period of 30hours, the material was dried by the circulation of hot dry air over itat a temperature which d'idnot permit the temperature of the culture toexceed 95 F. The preserved culture gave an obligate anaerobic bacteriacount of 3 billion per gram after storage for one month. The enzymeactivity of this material when tested on casein and starch was verygood.

EXAMPLE III A nutrient substrate was prepared and processed as inExample I except that the mixture after steaming was neutralized to pH7.0. The culture medium was then inoculated with a suspension preparedas follows:

300 lbs. Warm clean water 10 lbs. 24 hour broth culture B. licheniformis2 lbs. tomato juice 13 grams sodium bisulfide 1 lb. suspension of henfeces The mass was thoroughly and quickly mixed, then spread on trays ina layer about 1 /2 inches deep and placed in the incubator. Thetemperature of the material was maintained at 86 F. by live steam for aperiod of 18 hours. Limited aerobic conditions were maintained untilabout the 8th hour, when the agitation of the air was increased and thetemperature of the air so regulated that overheating did not take place.After the 18th hour, the live steam was cut off and the heat produced bygrowth of the culture utilized. After the 22nd hour, the culture reachedits zenith and at the 28th hour the culture was broken up and dryinginstituted by circulation of hotdry air at a temperature regulated tomaintain a maximum of 107 F. in the culture material. Drying wascomplete at hours. The dried culture showed an anaerobic bacteria countof 16 billion per gram.

EXAMPLE IV A culture particularly adapted to the treatment of sewage wasmade as follows:

A nutrient substrate composed of the following ingredients was prepared:

700 lbs. rice hulls 200 lbs. wheat bran 5 lbs. dried brewers yeast 20lbs. soybean meal 20 lbs. alfalfa meal lbs. dextrose 3 lbs. monocalciumphosphate /2 lb. trace mineral mixture 500 lbs. water 2500 lbs.commercial HCl The substrate was prepared and processed as in Example I,except that the mixture after steaming was neutralized to pH 7.0. Themixture was cooled to F. and inoculated with 300 lbs. of inoculating suspension prepared as follows:

300 lbs. warm clean water 10,lbs of wash. water containing washings andscrapings of :trick'ling filter 3 lbs. tomato juice 1 lb. sludge frombottom of trickling filter The mass was thoroughly and quickly mixed,then A spread on trays in a layer about 1 /2 inches deep and placed inthe incubator. The temperature of the material was maintained at 86 F.by live steam for a period of 18 hours. The air admitted to theincubator was diluted with CO2 until about the 8th hour, when theagitation of the air was. increased, and the temperature of the air soregulated that overheating did not take place. After the 18th hour, thelive steam was cut off and the heat produced by the actively growingculture utilized. After the 22nd hour, the culture reached its zenithand at the 28th hour the culture was broken up and drying instituted bymeans of hot, circulating air at a temperature controlled to maintainthe culture at a maximum of about 107 F. Drying was complete at 20hours. After drying, the aerobic bacteria was 2 billion and the obligateanaerobic count was 5 billion.

EXAMPLE V A culture particularly adapted for the treatment andconditioning of soil was made as follows:

A nutrient substrate composed of the following ingredients was prepared:

900 lbs. ground rice hulls 65 lbs wheatbran ZSlbs. molasses 5 lbs.monocalcium phosphate 2 lbs. potassium nitrate 3 lbs. ammonium sulfatelb. trace minerals 800 lbs. water The mixture wasadjusted to pH 6.2 withHCl without steaming or sterilizing, and then inoculated with 300 lbs.of the following suspension prepared as follows:-

300 lbs. clean warm water 5 lbs. of dry fungi growth containing sporesof Aspergillus,

Mucor, Rhizopus, Trichoderma and Penicillium 1 lb. soil containinganaerobic bacteria in rich humus.

The culture was mixed and spread on trays in a layer about 3 inches deepand allowed to incubate for 3 days under initially restricted andsubsequently increased aerobic conditions as described in the precedingexamples.

The black mat which formed throughout the material was broken up,moistened with 25 parts of water and neutralized with lime to pH 7. Itwas again inoculated with neutral soil containing soil nitrogen-fixingbacteria such as Rhizobium, Radiobacter, Azotobactor, Aerobactor andother heterotrophic bacteria together with various types of autotrophicmicroorganisms.

The mixture was incubated for 2 days. At the end of this time, itassumed a dark brown granular character with a pleasant, earthy odor.The mixture was then dried by means ofhot air regulated so that thetemperature of the culture did not exceed 100 F. A viable bacteria countshowed 6 billion obligate anaerobic bacteria per gram and 3V2 billionaerobic bacteria per gram.

EXAMPLE VI 9 was added to the substrate. The final culture. containedabout 6 billion anaerobic bacteria per gram.

The cultures produced according to my process may be utilized in manyways.

Because of the extremely high viable bacterial concentration, thecultures are particularly suitable for seeding or mass inoculation incommercial biological processes designed to produce desired associatedfactors such as B-complex vitamins, beneficial enzymes, growth-promotingfactors, antibiotics and the like. It is an established fact that, inmany cases, a few bacteria when introduced into a specific medium willnot produce the desired result. However, when large concentrations areused, they produce considerably larger amounts of the desired factorthan would normally be expected on the basis of the number of bacteriapresent. For example, Churchman and Kohn (Journal of ExperimentalMedicine, 1921, 33, 583) showed that although a single cell would notgrow in the presence of gentian violet, thirty cells could initiategrowth and accomplish much more than thirty times what one cell couldaccomplish.

Preserved, viable rumen cultures are especially valuable in the feedingof livestock, particularly of the ruminant varieties, since it serves tointroduce into the rumen desirable rumen organisms in sufficiently largenumbers effectively to establish or to supplement rumen activity at anoptimum level for the well-being of the animal.

Where suitable anaerobic organisms are cultured according to my process,as, for example, organisms obtained from rumen contents, hen feces,cow.manure and the like, considerable amounts of desirable associatedfactors, including B-complex vitamins, enzymes, unidentifiedgrowth-promoting factors, antibiotics and the like, are produced andpreserved in concentrated form in the desiccated cultures so that thepreserved cultures are particularly advantageous for use as supplementsboth for livestock feeding and human consumption. The B-complex vitaminsproduced in the cultures include B12, riboflavin, niacin, pantothenicacid and choline. Furthermore the ingested viable organisms themselvesproduce the desired factors in vivo.

Example VII clearly illustrates the large amounts of unidentifiedgrowth-promoting factors obtainable by' the use of such cultures. Thegrowth response in chicks, for example, is comparable to or evenhigherthan that obtained with other rich sources such as fish solublesand fishmeal.

EXAMPLE VII Tests were made to determine the growth response due tounidentified growth factors obtained by the use of the cultures preparedaccording to Examples I and III and also cultures of Aspergillus oryzaeand anaerobic organisms obtained from hen feces and cow manure.

The standard test procedure and basal ration employed were thoserecommended by the Wisconsin Alumni Research Foundation whereby thegrowth factors are determined by the added growth obtained when the testmaterial is fed to chicks in addition to a balanced ration containingall the known vitamins.

1 New Hampshire chicks-17 to the group.

Cultures may be prepared according to my. process which are especiallyvaluable for the digestion of 10 organic materials, These may be appliedin many spheres of industrial activity and are particularly effective inthe treatment of sewage and other industrial wastes such as petroleumwastes. The cultures may be tailored according to the particular use byproper selection of organisms.

The cultures employed in sewage disposal are effective both in aerobicphases of treatment as, for example, in the aerated, activated sludge ortrickling filter processes and in anaerobic phases such as septic tankor sludge digestion. In the former case the aerobic bacteria in theculture become active and in the latter case the anaerobes carry out thedigestion processes. The large concentrations of enzymes present in theseeding culture are also very important since they accelerateconsiderably the digestion processes. High concentrations of selectedbacteria in the seeding culture are particularly important in sewagedigestion in order to provide for vigorous growth of the desirableorganisms since, otherwise, there is a marked tendency for the digestingorganisms to be choked out by proliferation of undesirable contaminantsor destroyed by end products of such organisms.

EXAMPLE VIII A dried culture prepared as in Example II was employed inan effort to improve sewage digestion conditions in a sewage treatmentplant in Houston, Texas. The sludge digester was a reinforced concretetank with a circular fixed cover and a hopper bottom and had a capacityof 1200 cubic feet. For a two week period prior to use of the culture,the BOD averaged 1782 p. p. m., suspended solids averaged 1908 p. p. m.and pH averaged 5.9.

The culture was added to the contents of the digester first in a 10 lb.batch and for two weeks thereafter was added daily in 2 lb. quantities.The dosage was increased to 4 lbs. daily for 12 days and then raised to5 lbs. daily for two more weeks after which time it was discontinued.The BOD dropped to an average of 719 p. p. m. and suspended solids to328 p. p. m. The pH rose to 6.5. Conditions continued to improve for 3weeks after cessation of treatment and improved conditions were still ineffect 60 days after addition of the culture was discontinued. It wasalso noted that the quantity of sludge available for drawing dropped toless than 50% of the previous level.

EXAMPLE IX The culture prepared according to Example IV was mixed withequal parts of the dried culture prepared according to Example II. Thiswas introduced into a sludge lagoon of a plant in Texas that had apartially decomposed digester sludge. Within 12 hours after applicationof the culture, the lagoon became very active as indicated by a numberof gas bubbles emerging at the surface. All floating sludge sank to thebottom of the lagoon and within three days a layer of green algaecovered the entire lagoon with no odor being observed in the vicinity.The activity of the lagoon was then continuously maintained by theaddition of two pounds of the mixed culture into the wet Well of theplant between the pumpings of sludge to the lagoon.

EXAMPLE X A dried culture prepared according to Example V was tested forits soil enriching and conditioning properties as follows:

A clay soil of low humus content was sifted and thoroughly mixed withblue grass and clover seeds. The soil and seed mixture was then dividedinto 50 gram portions. The culture in varying concentrations was addedto several of the soil samples. Synthetic soil conditioners were addedto several other portions and one portion was re tained as a controlwith no additions.

Each soil portion Was placed in a cup 3 inches in diameter and having aperforated bottom. Equal amounts of Water were added daily to each ofthe petri dishes in which the cups had been placed. At the end of days,the control and each of the soii samples containing the syntheticconditioners had begun to dry out and crack. The portion containing aslittle as 0.1% of culture retained more moisture and portions containing0.4% showed maximum growth of grass and clover and no cracking.

Although this invention has been described with reference toillustrative embodiments thereof, it will be apparent to those skilledin the art that modifications may be made and that the principles of theinvention may be embodied in other forms but within the scope of theinvention and of the appended claims.

I claim:

1. A process for culturing anaerobic bacteria which comprises admixingobligate anaerobic bacteria with obligate aerobic vegetativemicroorganisms, the aerobic organisms being characterized by the abilityactively to absorb oxygen and to grow rapidly, and incubating themixture of organisms on a nutrient substrate in the presence of air,said air being present throughout the incubating period to provide forthe growth of the aerobic organisms.

2. A process for culturing anaerobic bacteria which comprises admixingobligate anaerobic bacteria with aerobic mold, the mold beingcharacterized by the ability actively to absorb oxygen and to growrapidly, and incubating the mixture of organisms on a nutrient substratein the presence of air, the amount of air in the incubating chamberbeing initially limited and being increased as the aerobic organismsincrease in growth.

3. A process for culturing anaerobic bacteria which comprises admixingobligate anaerobic bacteria with obligate aerobic bacteria, the aerobicbacteria being characterized by the ability actively to absorb oxygenand to grow rapidly, and incubating the mixture of organisms on. anutrient substrate in the presence of air, the amount of air in theincubating chamber being initially limited and being increased as theaerobic organisms increase in growth.

4. A process for culturing and preserving anaerobic bacteria whichcomprises admixing obligate anaerobic bacteria with obligate aerobicbacteria, the aerobic bacteria being characterized by the abilityactively to absorb oxygen, to grow rapidly and to maintain a minimum pHof about 5.5, and incubating the mixture of organisms on a granularnutrient substrate in the presence of air, the amount of air in theincubating chamber being initially limited and being increased as theaerobic organisms increase in growth, and then drying the culture bycirculating hot dry air at a temperature controlled to maintain thetemperature of the culture below the thermal death point of themicroorganisms to form a desiccated culture containing viable aerobicand obligate anaerobic bacteria.

5. A process for culturing and preserving anaerobic bacteria whichcomprises admixing obligate anaerobic bacteria with aerobic mold, themold being characterized by the ability actively to absorb oxygen and togrow rapidly, and incubating the mixture of organisms on a granularnutrient substrate in the presence of air, the amount of air in theincubating chamber being initially limited and being increased as theaerobic organisms increase in growth, and then drying the culture bycirculating hot, dry air at a temperature controlled to maintain thetemperature of the culture below the thermal death point of themicroorganisms to form a desiccated culture containing viable aerobicand obligate anaerobic microorganisms.

6. A process for culturing and preserving anaerobic bacteria, whichcomprises admixing obligate anaerobic bacteria with obligate aerobicbacteria, the aerobic bacteria being characterized by the abilityactively to absorb oxygen, to grow rapidly and to maintain a pH of about6.5 to alkaline, incubating the mixture of organisms on a granularnutrient substrate in the presence of air, the monster air in theincubating chamber being initially limited and being increased theaerobic organisms increase in growth, at atemperature such that thetemperature of the culture does not exceed a maximum of about F., andthen drying the culture by circulating hot dry air at a temperaturecontrolled to maintain the temperature of the culture at a maximum ofabout 107 F. to form a desiccated culture containing viable aerobic andobligate anaerobic bacteria.

7. A process for culturing and preserving anaerobic bacteria whichcomprises admixing obligate anaerobic bacteria with mold selected fromthe group consisting of Aspergillus, Penicillium, Rhyzopus, Trichodermaand Mucor, incubating the mixture of organisms on a nutrient substratehaving a pH in the range of about 6.0 to 7.0 in the presence of air, theamount of air in the incubating chamber being initially limited andbeing increased as the aerobic organisms increase in growth, at atemperature such that the temperature of the culture does not exceed amaximum of about 105 F., and then drying the culture by circulating hot,dry air at a temperature controlled to maintain the temperature oftheculture at a maximum of about 107 FL to form a desiccated culturecontaining viable aerobic and obligate anaerobic microorganisms.

8. A process for culturing and preserving anaerobic bacteria whichcomprises admixing obligate anaerobic bacteria with aerobic bacteriaselected from the genus Bacillus, family Bacillaceae, the aerobicbacteria being characterized by the ability actively to absorb oxygen,to grow rapidly and to maintain a pH from about 6.5 to alkaline,incubating the mixture of organisms on a granular nutrient substratehaving a pH in the range of about 6.0 to 7.0 in the presence of air, theamount of air in the incubating chamber being initially limited andbeing increased as the aerobic organisms increase in growth, at atemperature such that the temperature of the culture does not exceed amaximum of about 105 F., and then drying the culture by circulating hotdry air at a temperature controlled to maintain the temperature of theculture at a maximum of about 107 F. to form a desiccated culturecontaining viable aerobic and obligate anaerobic bacteria. A

9. A process for culturing and preserving anaerobic rumen organismswhich comprises admixing the organisms obtained from the rumen of aruminant animal including obligate anaerobic rumen bacteria with aerobicmold, the mold being characterized by the ability actively to absorboxygen and to grow rapidly, incubating the mixture of organisms on agranular nutrient substrate in the presence of air, the amount of air inthe incubating chamber being initially limited and being increased asthe aerobic organisms increase in growth, at a temperature such that thetemperature of the culture does not exceed a maximum of about 105 F.,and then drying the culture by circulating hot dry air at a temperaturecontrolled to maintain the temperature of the culture at a maximum ofabout 107 F. to form a desiccated culture containing viable obligateanaerobic rumen organisms and aerobic mold.

10. A process for culturing and preserving anaerobic rumen organismswhich comprises admixing the organisms obtained from the rumen of aruminant animal including obligate anaerobic rumen bacteria withAspergillis oryzae, incubating the mixture of organisms on a granularnutrient substrate having a pH in the range of about 6.0 to 7.0 in thepresence of air, the amount of air in the incubating chamber beinginitially limited and being increased as the aerobic organisms increasein growth, at a temperature such that the temperature of the culturedoes not exceed a maximum of about 105 F., and then drying the cultureby circulating hot dry air at a temperature controlled to maintain thetemperature of the culture at a maximum of about 107 F., to form adesiccated culture containing viable obligate anaerobic rumen organismsand Aspergillis oryzae.

11. A process for culturing and preserving aerobic and anaerobicbacteria suitable for the treatment of sewage, which comprises admixingobligate anaerobic bacteria which are characterized by their ability todigest organic matter with obligate aerobic vegetative microorganisms,the aerobic organisms being characterized by the ability actively toabsorb oxygen and to grow rapidly, incubating the mixture of organismson a granular nutrient substrate in the presence of air, the amount ofair in the incubating chamber being initially limited and beingincreased as the aerobic organisms increase in growth, at a temperaturesuch that the temperature of the culture does not exceed a maximum ofabout 105 F., and then drying the culture by circulating hot, dry air ata temperature controlled to maintain the temperature of the culture at amaximum of about 107 F. to form a desiccated culture containing viableaerobic obligate anaerobic microorganisms.

References Cited in the file of this patent UNITED STATES PATENTS OTHERREFERENCES Hoover et al.: Sewage and Industrial Wastes, January 1952,pp. 38-43.

1. A PROCESS FOR CULTURING ANAEROBIC BACTERIA WHICH COMPRISING ADMIXINGOBLIGATE ANAEROBIC BACTERIA WITH OBLIGATE AEROBIC VEGETATIVEMICROORGANISMS, THE AEROBIC ORGANISMS BEING CHARACTERIZED BY THE ABILITYACTIVELY TO ABSORB OXYGEN AND TO GROW RAPIDLY, AND INCUBATING THEMIXTURE OF ORGANISMS ON A NUTRIENT SUBSTRATE IN THE PRESENCE OF AIR,SAID AIR BEING PRESENT THROUGHOUT THE INCUBATING PERIOD TO PROVIDE FORTHE GROWTH OF THE AEROBIC ORGANISMS.